Identification of C-Src Tyrosine Kinase Substrates Using Mass

Identiﬁcation of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Ramars Amanchy,† Jun Zhong,† Henrik Molina,†,‡ Raghothama Chaerkady,†,# Akiko Iwahori,† Dario Eluan Kalume,†,⊥,§ Mads Grønborg,† Jos Joore,§ Leslie Cope,| and Akhilesh Pandey*,†

McKusick-Nathans Institute of Genetic Medicine and the Departments of Biological Chemistry, Pathology and Oncology, Johns Hopkins University, Baltimore, Maryland 21205, Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Center for Experimental Bioinformatics, Odense 5230, Denmark, Pepscan Systems, Edelhertweg 15, 8219 PH Lelystad, The Netherlands, and Sidney Kimmel Comprehensive Cancer Center and the Department of Biostatistics, Bloomberg School of Public Health, and Johns Hopkins University, Baltimore, Maryland 21205

Received March 17, 2008

c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues that are phosphorylated by c-Src on the novel c-Src substrates, we designed custom peptide microarrays containing all possible tyrosine-containing peptides (312 unique peptides) and their mutant counterparts containing a Tyr f Phe substitution from 14 of the identified substrates. Using this platform, we identified 34 peptides that are phosphorylated by c-Src. We have demonstrated that SILAC-based quantitative proteomics approach is suitable for identification of substrates of nonreceptor tyrosine kinases and can be coupled with peptide microarrays for high-throughput identification of substrate phosphopeptides.

Keywords: Phosphorylation • SILAC • PDGF • Quantitative mass spectrometry • Systems biology

Introduction and cell survival.1 One vital step in understanding the role of c-Src kinase in cellular transformation and signaling is sys- Most signaling pathways include protein kinases and their tematic identification of all of its potential cellular substrates substrates that serve as means to amplify signals from extra- involved in these processes. cellular signals and other stimuli. However, the precise con- Recent studies based on advances in mass spectrometry- nectivity between protein kinases and their downstream sub- based proteomics have provided large-scale catalogs of phos- strates has not been fully elucidated for most protein kinases. phorylation sites.2–5 However, determination of kinases re- c-Src is a classic nonreceptor tyrosine kinase that has been implicated in regulation of cytoskeletal rearrangement and cell sponsible for these phosphorylation events is not an easy task adhesion networks that control cell migration, cell proliferation owing to the transient interaction between kinases and their substrates. Chemical and genetic approaches have been previ- * Corresponding author: Tel, 410-502-6662; fax, 410-502-7544; e-mail, ously used to identify c-Src substrates. Such studies include [email protected]. † use of an ATP analogue that is a specific substrate for an McKusick-Nathans Institute of Genetic Medicine and the Departments 6 of Biological Chemistry, Pathology and Oncology, Johns Hopkins University. analogue-specific allele of v-Src, screening of cDNA expression ‡ University of Southern Denmark. libraries with anti-phosphotyrosine antibodies7 and use of ⊥ Current address: Department of Tropical Medicine, Oswaldo Cruz mutant inducible forms of c-Src.8 To date, several c-Src Foundation - FIOCRUZ, Rio de Janeiro, RJ, 21040-900, Brazil. § Pepscan Systems. substrates as well as interactors have been reported. Human | Sidney Kimmel Comprehensive Cancer Center and the Department of Protein Reference Database (HPRD)9 provides a list of 132 c-Src Biostatistics, Bloomberg School of Public Health, and Johns Hopkins University. mediated phosphorylation sites in 64 known substrates along # Institute of Bioinformatics, International Technology Park. with 204 proteins that interact with c-Src.

3900 Journal of Proteome Research 2008, 7, 3900–3910 10.1021/pr800198w CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008 Identification of c-Src Kinase Substrates research articles We have used the stable isotope labeling with amino acids Immunoprecipitation and Western Blotting. Light, medium in cell culture (SILAC) approach which enables identification and heavy cell lysates were precleared with protein A-agarose, of tyrosine kinase substrates based on a unique signature in mixed, and incubated with 400 µg of 4G10 monoclonal anti- mass spectrometry experiments.10–12 The main objective of this bodies coupled with agarose beads, 75 µg of biotin-conjugated work was to identify novel c-Src substrates by overexpression RC20 antibody, and streptavidin-agarose beads overnight at 4 of a constitutively active form of c-Src followed by enrichment °C. Precipitated immune complexes were then washed three of tyrosine-phosphorylated proteins. We have identified 26 times with lysis buffer. Agarose beads were boiled and resolved novel c-Src tyrosine kinase substrates in addition to 10 others, by 10% SDS-PAGE. The gel was silver-stained for visualizing which were either known Src family kinase substrates or protein bands. Western blotting experiments were performed proteins known to associate with Src family kinases. We have using anti-phosphotyrosine antibody (4G10) and reprobing was experimentally confirmed 4 novel substrates, NICE-4, RNA carried out using anti-Flag antibody. binding motif 10, FUSE-binding protein 1 and TRK-fused gene, Cloning and Transfection. NICE-4 protein (NP_055662), to be direct substrates of c-Src using in vitro kinase assays. We RNA Binding Motif protein 10 isoform 1 (NP_05667), Far were also able to implicate EWS1, RNA binding motif 10 and upstream element-binding protein (NP_003893), and TRK- Bcl-2 associated transcription factor in PDGF signaling using Fused gene (NP_006061) were subcloned into a Flag epitope- a chemical inhibitor of c-Src. Our peptide microarray approach tagged mammalian expression vector, pCMVtag4A. 293T cells led to identification of a number of peptides that are phos- were grown in 10 cm dishes. One dish transfected with wild- phorylated by c-Src. To our knowledge, this is the first reported type c-Src and pCMVtag4A vector as control; one was cotrans- integrated proteomics strategy that couples cell culture, mass fected with wild-type c-Src and Flag-tagged cDNAs. The spectrometry and peptide microarrays to identify tyrosine expressed proteins were immunoprecipitated using anti-Flag kinase substrates. antibody, followed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine antibody followed Experimental Procedures by stripping and reprobing with anti-Flag antibodies. Chemicals and Antibodies. Stable isotope containing amino In Vitro Kinase Assays Using GST-Fusion Proteins. Fusion 12 13 13 15 proteins were made using TNT-coupled rabbit reticulocyte acids, C6-arginine, C6-arginine and C6- N4-arginine, were purchased from Cambridge Isotope Labs (Andover, MA). lysate system (Promega, Madison, WI) with the cDNAs cloned Complete protease inhibitor cocktail tablets were purchased in GST expression vector, PGEX4T1. The in vitro translated from Roche (Indianapolis, IN), sodium orthovanadate and anti- GST-tagged proteins were purified with 10 µg of GST beads for ° Flag M2 monoclonal antibody from Sigma-Aldrich Co. (St. 12hat4 C. After incubation, the beads were washed two times Louis, MO), SU6656 from EMD Biosciences, Inc. (San Diego, in lysis buffer and two times in kinase buffer (20 mM Hepes, CA), anti-phosphotyrosine antibodies (4G10) agarose-conjugate pH 7.4, 5 mM MgCl2, 2 mM MnCl2,50µM sodium vanadate, and streptavidin-agarose beads from Upstate Biotechnology 50 µM DTT). Immune complexes were incubated for 30 min ° (Lake Placid, NY), antiphosphotyrosine-RC20 biotin conjugate at 30 Cin5µL of ATP mixture (10 µM cold ATP and 10 µCi of 32 from BD Biosciences (San Jose, CA) and PDGF-BB from [γ- P] ATP) and c-Src Kinase. Protein samples were then eluted Invitrogen (Carlsbad, CA). Sequencing grade trypsin was pur- by boiling in sample buffer and resolved by SDS-PAGE. The 32 chased from Promega (Madison, WI). Antibodies against cort- gel was dried and exposed to X-ray film to visualize the P- actin were purchased from Upstate USA, Inc. (Chicago, IL), labeled protein bands. p130CAS and EWS1 from Santa Cruz Biotechnology, Inc. (Santa PDGF Stimulation and Inhibition of Src Kinase. NIH3T3 Cruz, CA), BTF from Bethyl, Inc. (Montgomery, TX), and RBM10 cells were grown in DMEM containing 10% FBS supplemented was from Abcam, Inc. (Cambridge, MA). Phospho-Src (Tyr416) with antibiotics. For all PDGF stimulation experiments, cells antibody and PhosphoScan Kit (P-Tyr-100) were purchased were stimulated with 100 ng/mL PDGF-BB for 5 min. For from cell signaling technology (Boston, MA). inhibition of c-Src kinase, cells were treated with 2 µM c-Src Cell Culture and Stable Isotope Labeling with Amino kinase inhibitor, SU6656, for 1 h prior to stimulation of cells Acid in Cell Culture (SILAC). Human embryonic kidney 293T with PDGF-BB for 5 min. cells were grown in Dulbecco’s modified Eagle’s medium In-Gel Trypsin Digestion and In-Solution Trypsin Diges- (DMEM) containing ‘light’, ‘medium’ or ‘heavy’ arginine supple- tion. The silver-stained protein bands were excised and in-gel mented with 10% dialyzed fetal bovine serum (FBS) plus trypsin digestion was performed as described previously.15 antibiotics. The 293T cells were adapted to growing in isotope Briefly, the gel slices were excised and incubated with trypsin rich-medium supplemented with dialyzed serum prior to overnight at 37 °C to allow digestion of proteins after a initiating these experiments. In each experiment, 20 10-cm reduction and alkylation step. After in-gel digestion, the tryptic dishes were used per condition and the cells were transfected peptides were extracted. The supernatants from the in-gel and with 15 µg of DNA using the standard calcium phosphate in-solution trypsin digestion containing the peptide mixture method (Invitrogen, Carlsbad, CA). Six hours after transfection, were partially dried down in a vacufuge to approximately 10 the cells were serum-starved for 10 or 20 h. After starvation, µL. For in-solution digestion and enrichment of phosphopep- the cells were lysed in modified RIPA buffer (50 mM Tris-HCl, tides, phosphoscan kit was used according to manufacturer’s pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% prescribed conditions. sodium deoxycholate, and 1 mM sodium orthovanadate in the Liquid Chromatography-Mass Spectrometry. The extracted presence of protease inhibitors). Upon cell lysis, proteins lysates peptide mixture was centrifuged for 2 min at 12 000g and 4 °C were either subjected to affinity purification of tyrosine phos- and resolved by reversed-phase liquid chromatography on phorylated proteins13 or peptides containing phosphotyrosine Agilent 1100 Series LC system (Agilent Technologies, Palo Alto, were enriched directly from trypsin-digested cell lysates14 using CA) equipped with a well plate sampler, a vacuum degasser, specific antibodies against phosphotyrosine and identified by and a capillary pump. Each fraction from the digested peptide tandem mass spectrometry. mixture was analyzed by automated nanoflow LC-MS/MS. An

Journal of Proteome Research • Vol. 7, No. 9, 2008 3901 research articles Amanchy et al. Agilent Technologies 1100 series system was used to deliver a carried out using these custom peptide microarrays by incu- flow of 1.5 µL/min during desalting of the sample and 250 nL/ bating 50 ng of recombinant c-Src Kinase (Invitrogen, Carlsbad, min during elution of the peptides into the mass spectrometer CA) in kinase reaction buffer and 300 µCi/ml γ33P-labeled ATP as described before.10 Each sample was loaded onto an online (AH9968; GE Healthcare Biosciences Corp., Piscataway, NJ) at analytical fused silica needle column (Proxion Biosystems, 25 °Cfor1hina120µL reaction volume supplemented with Odense, Denmark) packed with 5-µm Vydac C18 resin. Washing 200 µM ATP. The reaction was stopped and the following and desalting was done with 95% mobile phase A (H2O with washing steps were performed: 2 washes in 2 M sodium 0.4% acetic acid and 0.005% heptafluorobutyric (v/v)) and 5% chloride containing 1% Triton X-100 followed by 3 washes in mobile phase B (90% acetonitrile, 0.4% acetic acid, 0.005% phosphate buffered saline containing Triton X-100 and 1 wash heptafluorobutyric acid in water). Samples were eluted from in distilled water. The glass slides were then air-dried and the analytical column by a linear gradient of 90% mobile phase exposed to the phosphorimager screen for 12 h and scanned A to 60% mobile phase A. A 34-min gradient was used for using Biorad Molecular Imager FX (Bio-Rad Laboratories, Inc., elution. A potential of 2.8 kV was applied to the emitter (Proxion Hercules, CA). The image was processed using GenePix Pro 6.0 Biosystems). The spectra were acquired on a quadrupole time- software (Molecular Devices Corporation, Sunnyvale, CA). of-flight mass spectrometer (Q-TOF US-API, Micromass, Autoradiographs were obtained using a phosphorimager screen. Manchester, U.K.) equipped with an ion source sample intro- The assay was performed in triplicate. The intensity values duction system designed by Proxeon Biosystems (Odense, obtained were transformed to the log base 2 scale. Effects on Denmark). Data were obtained in positive ion mode. Data- intensity due to the position of the spot on the slide were dependent acquisition was performed with a ion mass window estimated by performing a local regression analysis (loess) with to 2.5 Da. MS to MS/MS switch was set to a threshold of 10 respect to chip coordinates, and subtracted out.19 Normalized counts/s, and MS/MS to MS was set to an intensity below a log 2 intensities for triplicate spots were averaged, and the threshold of 2 counts/s. Charge state recognition was used to mean log 2 MUT intensity was subtracted from the corre- estimate the collision energy for the fragmented precursor. Scan sponding mean log 2 WT intensity for each peptide. The time was set to 0.9 s, and interscan time was set to 0.1 s. The resulting background adjusted values were averaged over number of components (i.e., number of MS/MS per MS scan) replicate arrays. The mean background adjusted log intensity was set to three resulting in a total cycle time (one MS and for each peptide on the Y-axis, and the average of WT, and three MS/MS spectra) of 10 s. The acquisition of data was MUT log intensities on the X-axis were plotted to estimate the performed using MassLynx (version 4.0). The parameters used distribution of the intensity values arising from the phospho- for generating peak lists from the raw data were the following: rylated peptides. A key assumption for selection of positive smooth window, 4.00; number of smooth, 2; smooth mode, (phosphorylated) peptides is that WT peptide intensity values Savitzky Golay; and percentage of peak height to calculate are greater than MUT peptide intensities. It is also assumed centroid spectra, 80% with no baseline subtraction. The gener- that higher intensity peptides are more likely to be positive. ated peak lists (pkl-file) were searched against the RefSeq We note that, consistent with this assumption, the WT intensity human protein database (build 33, 29 572 sequences) (ww- is consistently higher than MUT intensity for the high intensity w.ncbi.nlm.nih.gov./RefSeq/) using Mascot version 2.0, with a peptides on the right side of the plot. Likewise, there is greater mass accuracy of 1.1 Da for the parent ion (MS) and 0.2 for symmetry on the left side quadrant of the MvA plot, where we the fragment ions (MS/MS), allowing a maximum of two missed expect nonphosphorylated peptides to have WT intensities that cleavages. Carbamidomethylation of cysteines was considered are as likely to be lower than MUT values as higher. The as fixed modification, and oxidations of methionine residues, classical False Positive Rate (expected error rate for a set of “medium” arginine (+6 Da), “heavy” arginine (+10 Da), and points) is derived by evaluating symmetry in the Y-axis. For phosphorylation of tyrosine residue were considered as variable each value of A, the classical FPR blue curve gives: (number of modifications. An initial protein list was generated using the points below the X-axis to the right of A)/(number of points following criteria. Only proteins containing at least one unique above the X-axis to the right of A), and describes the expected peptide (if the sequence has not been assigned to a different number of false positives in the upper right quadrant of the protein) with a Mascot score over 30 were considered in the plot. Sometimes it is desirable to estimate the probability that data set. The sequence of higher scoring peptides was manually a single given point is a false positive, allowing us to move the verified. Quantitation was performed on three to four peptides threshold to the left until the price of adding one more peptide (wherever available) by comparing the extracted ion chromato- is too high. This is described by the local false positive rate gram of the corresponding light and heavy peptides using MS- curve. The local FPR curve gives the probability of a positive Quant.16 Reproducibility of measurements was performed by peptide located at A being a false positive. We selected all using two analysis of variance models as described earlier.17 peptides where the local false positive rate was lower than 0.15 The tandem mass spectra were manually verified to assign the and WT intensities were 2-fold greater compared to MUT. The sequence and phosphorylation sites for all peptides identified resulting set has an overall FPR of 0.085. in this study (Supplementary Table 1). The phosphorylation sites were manually verified and assigned after confirmation Results and Discussion of a mass difference of 243 Da corresponding to phosphotyrosine residue. Stable Isotope Labeling of Cellular Proteins for the Iden- Peptide Microarrays and Data Analysis. The WT (peptides tification of c-Src Kinase Substrates. SILAC involves metabolic containing a tyrosine residue in the center) and MUT (peptides labeling of cellular proteomes by growing the cells in media where the central tyrosine residue was replaced by phenyla- containing amino acids labeled with stable isotopes. SILAC lanine) peptides (Supplementary Table 2) were each spotted enables identification of peptides labeled in vivo and relative as triplicates on glass slides (Pepscan Systems, Lelystad, quantitation of abundance of the peptides arising out of a Netherlands) as described earlier.18 c-Src kinase assays were mixture of labeled and unlabeled protein samples.15 This

3902 Journal of Proteome Research • Vol. 7, No. 9, 2008 Identification of c-Src Kinase Substrates research articles stitutively active form of c-Src for 24 h (Figure 2). We have also observed that the Y416 in c-Src gets phosphorylated more in this state which points to the increased c-Src tyrosine kinase activity (Figure 2). In addition, the trend of increasing tyrosine phosphorylation could also serve as one more surrogate signature of substrates as one would expect the phosphorylation level to increase during this time course. Mixing of light, medium and heavy isotope labeled cell lysates allowed us to compare the profile of proteins in a single MS experiment. In MS/MS spectra, fragmentation patterns generated by light, medium and heavy peptide pairs are identical except for the expected mass shift of the fragment ions. The ratio of the intensity of the heavier versus the light peptides provides information about the degree of phosphorylation of a protein and hence its enrichment upon expression of an active c-Src kinase. Thus, the greater the extent of phosphorylation of a protein by c-Src kinase, the higher should be its abundance in anti-phosphotyrosine antibody immunoprecipitates. Peptide sets with a little or no increase in intensity indicate that the protein is not different in abundance in the different states being compared. Such proteins were not Figure 1. (A) Schematic for the integrated proteomic approach investigated further as they are likely nonspecifically bound for the identification of c-Src kinase substrates. Human embry- proteins. An increase in heavy/light intensity ratio, indicating onic kidney (HEK) 293T cells growing in Arg ‘0’ containing an increase in total phosphotyrosine content upon Src kinase medium were transiently transfected with a kinase-dead Src expression and activity, was found in peptides derived from (K298M) and 293T cells growing in Arg ‘6’ and Arg ‘10’ were 36 proteins (Tables 1 and 2). Of these, 10 proteins were either transiently transfected with constitutively active Src kinase known Src family kinase substrates or proteins known to 12 13 (Y527F). Arg ‘0’ refers to C6-arginine, Arg ‘6’ refers to C6- interact with Src family kinases (Table 1), whereas the remain- arginine and Arg ‘10’ refers to 13C -15N -arginine, isotopic labeled 6 4 ing 26 proteins have not previously been described as sub- forms of arginine used to differentially label 293T cells for strates of c-Src or Src family members (Table 2) in higher identification of Src substrates. eukaryotes. The known substrates identified in this screen included EWS1 (Ewing sarcoma breakpoint region 1),27 cort- 28 29 method also allows one to distinguish contaminating proteins actin, calponin-3, hnRNP-K (Heterogeneous nuclear ribo- 30,31 in immunoprecipitates that arise due to nonspecific binding. nucleoprotein K), G3BP (RasGAP SH3-domain binding 32–34 35 Because of the complexity of cell lysates, and because kinase protein) and c-Src itself. The protein with maximum substrates generally exhibit low stoichiometry of tyrosine increase in tyrosine phosphorylation upon c-Src overexpression phosphorylation, specific identification of tyrosine kinase was c-Src itself. Other known and novel Src family substrates substrates can be facilitated by prior enrichment of tyrosine- displayed >2-fold increase in intensity of phosphorylation. phosphorylated proteins with anti-phosphotyrosine antibodies. Apart from signaling and cytoskeletal proteins, we also identi- We applied SILAC for the identification of the c-Src kinase fied DNA and RNA binding proteins in our analysis. Some of substrates in human embryonic kidney cells by overexpression the reasons it is not possible in a kinase-substrate identifica- of a constitutively active form of c-Src followed by affinity tion screen of this type to possibly identify every known purification of tyrosine-phosphorylated proteins. We transiently substrate are (i) previously described substrates might not be overexpressed either a kinase inactive c-Src (K298M) as a expressed in the cell line that we have used; (ii) although the control or a constitutively active c-Src (Y527F) kinase in 293T substrates might be expressed, they might not be abundant cells. Phosphorylation of the C-terminal tyrosine by C-terminal enough to be enriched and detected in our experiments; (iii) Src Kinase (CSK) allows inactivation of c-Src.20–22 Hence, the phosphorylation might not occur or occur at a lower level mutation of this tyrosine residue to phenylalanine allows c-Src in the cells that we have used; and (iv) the time course and kinase to be constitutively active by preventing its folding and kinetics of phosphorylation in the system that we have em- by allowing the kinase domain access to its substrates.23,24 ployed might be different from the systems previously used in Inhibition of c-Src activity is often achieved by coexpression the literature to describe substrates. Thus, although it is not of the c-Src-inactivating C-terminal Src Kinase (CSK), the possible to identify all of the known substrates of Src, identi- kinase-inactive Src mutant Src K298 M,25 or by treatment of fication of 6 known substrates of Src along with validation of the cells with c-Src inhibitors. some novel ones is indicative of the success of this type of Three populations of human embryonic kidney cells were phosphoproteomic screen. 12 13 grown in DMEM containing C6-arginine (light), C6-arginine We performed relative quantitation of tyrosine phosphory- 13 15 (medium) and C6- N4-arginine (heavy), respectively (Figure lation for each protein as a measure of increase in intensity 1). The cells grown in light medium were transfected with a ratios from light isotope to heavier isotope containing peptides kinase inactive c-Src as a negative control.26 Cells grown in (Supplementary Table 1). Figure 3 shows representative MS medium and heavy isotope containing media were transfected spectra for six of the proteins identified from our screen. In with a constitutively active form of c-Src (Y527F) and harvested addition to identification of proteins with increased phospho- at 12 or 24 h post-transfection, respectively. We found an rylation and quantitation, we also mapped 6 phosphorylation increased tyrosine phosphorylation upon transfection of con- sites; Y334 in cortactin10 (Figure 4A) and Y72 in hnRNP-K30

Journal of Proteome Research • Vol. 7, No. 9, 2008 3903 research articles Amanchy et al.

Figure 2. Tyrosine phosphorylation proﬁle of proteins on transfection with inactive and active forms of c-Src. 293T cells were transfected with inactive and active forms of c-Src, cells were lysed, and tyrosine-phosphorylated proteins were immunoprecipitated from the cell lysates as described in Experimental Procedures. Cell lysates and immunoprecipitates were then run on a 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with anti-phosphotyrosine antibodies and reprobed with phospho (Y416)- Src antibody.

Table 1. Known Src Family Kinase Substrates and Interactors Identiﬁed in This Study by Overexpression of c-Src Kinase in 293T Cells Followed by SILAC

NCBI accession no. protein fold increase (heavy/light)a ( SD Reference Known c-Src Substrates 1 NP_005408 c-Src 30 ( 9.1 35 2 NP_005222 Cortactin 4.9 ( 2.6 28 3 NP_005234 Ewing sarcoma breakpoint region 1 4.6 27 4 NP_112552 Heterogeneous nuclear ribonucleoprotein K 3.7 ( 0.2 30, 31 5 NP_001830 Calponin 3 3.4 29 6 NP_005745 RasGAP SH3-domain binding protein 2.2 32–34 Known Src Family Kinase Interactors 7 NP_112533 Heterogeneous nuclear ribonucleoprotein A2/B1 10.2 ( 5.1 45 8 NP_002129 Heterogeneous nuclear ribonucleoprotein D 4.3 45 9 NP_006833 Splicing factor 3B subunit 2 5.6 ( 0.9 46 10 NP_005511 Heterogeneous nuclear ribonucleoprotein H1 3.0 45

a Heavy refers to Arg ‘10’ and light refers to Arg ‘0’ containing peptides.

(Figure 4B) identified in this study were reported earlier. We do not know the reasons for such a low yield. However, a also identified 4 novel tyrosine phosphorylation sites on each protein IP serves our purpose of identifying the proteins that on EWS1 (Figure 4C), FUSE-binding protein 1 (Figure 4D), are substrates of Src even though the site is still not identified. calponin-3 (Figure 4E) and FIP1-like1 (Figure 4F). We note that, This is the reason we coupled our approach with peptide although EWS1 was a known c-Src substrate, no tyrosine microarrays to aid in identification of phosphopeptides. phosphorylation sites were previously localized. Validation of a Subset of Novel c-Src Substrates. Further One of the drawbacks of our experiments is that lysine was validation of the proteins identified by SILAC to prove that they not available as 3 different isotopic forms at the time we are bona fide substrates usually involves the use of antibodies initiated our experiments. By using 3 isotopes of lysine along against these proteins. The validation of all of the protein with arginine, we would have obtained a better peptide candidates is not always possible, especially for novel proteins, coverage for each protein and likely identified additional as it depends on the availability of good antibodies. As proteins as substrates of c-Src. Nevertheless, we performed a commercial antibodies were not available for many of the lysine and arginine labeled SILAC experiment in a similar proteins identified, we chose to investigate a subset of proteins, manner but followed by enrichment of phosphopeptides using if they were direct substrates of c-Src using in vitro kinase antibodies against phosphotyrosine to see if we could identify assays. We selected NICE-4, RBM10, FBP1 and TRK-fused gene phosphorylated peptides, but unfortunately, we could only for this purpose, as their cDNAs were readily available. We used identify 8 tyrosine phosphorylation sites (data not shown). We rabbit reticulocytes to perform in vitro transcription and

3904 Journal of Proteome Research • Vol. 7, No. 9, 2008 Identiﬁcation of c-Src Kinase Substrates research articles Table 2. List of Novel Potential Src Substrates Identiﬁed Using SILAC by Overexpression of Src Kinase in 293T Cells

accession no. protein fold increase (heavy/light)a ( SD NP_005454 Heterogeneous nuclear ribonucleoprotein D-like 10.0 ( 5.2 NP_113680 RNA binding motif protein 4B 9.4 ( 3.7 NP_003760 Splicing factor, arginine/serine-rich 9 9.3 ( 3.6 NP_004490 Heterogeneous nuclear ribonucleoprotein AB 8.0 NP_005667 RNA binding motif protein 10 7.2 ( 0.6 NP_003893 FUSE binding protein 6.9 ( 0.8 NP_055662 NICE-4 6.6 ( 2.1 NP_919223 Heterogeneous nuclear ribonucleoprotein A3 6.1 ( 2.5 NP_003676 FUSE binding protein 2 5.7 ( 2.2 NP_004951 FUS/TLS oncogene 5.0 ( 0.9 NP_005849 A-kinase anchor protein 8 5.5 NP_055554 Bcl-2-associated transcription factor 1 5.5 NP_877952 Arsenate resistance protein ARS2 5.4 NP_473357 FUS interacting protein (serine/arginine-rich) 1 5.1 NP_005110 Thyroid hormone receptor associated protein 3 5.1 NP_008937 Cleavage and polyadenylation specific factor 5 4.8 ( 3.1 XP_028253 Similar to Zinc finger CCCH-type domain-containing protein 6 3.8 ( 1.9 NP_079222 NEFA-interacting nuclear protein 3.8 ( 0.8 NP_060082 Zinc finger, CCHC domain containing 8 3.7 NP_005057 Splicing factor proline/glutamine-rich 3.1 ( 1.0 NP_005780 Proteasome activator subunit 3 2.6 ( 0.4 NP_006061 TRK-fused gene 2.4 NP_003746 Eukaryotic translation initiation factor 3, subunit 4 2.2 NP_663760 Ataxin 2 related protein 2.2 NP_067038 Chromosome 20 open reading frame 77 2.0 ( 0.3 NP_057123 Homeobox prox 1 2.0 ( 0.3

a Heavy refers to Arg ‘10’ and light refers to Arg ‘0’ containing peptides. translation reactions. These proteins were purified and incu- of a c-Src kinase inhibitor, SU6656 (2-oxo-3-(4,5,6,7-tetrahy- bated along with c-Src kinase to investigate if it could phos- dro-1 H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic phorylate these proteins. After incubation with c-Src kinase, acid dimethylamide).38 By activating PDGF signaling in NIH3T3 the proteins were resolved by SDS-PAGE and autoradiographs cells, we investigated the ability of these proteins to get were obtained. We found that all of the tested proteins were tyrosine-phosphorylated upon ligand-induced stimulation of tyrosine phosphorylated upon incubation with c-Src kinase the PDGF receptor. We found that all of the novel proteins were (Figure 5A). TRK-fused gene from Xenopus laevis has been tyrosine-phosphorylated upon stimulation of PDGF receptor shown to interact with SH3 domains of various proteins (Figure 5C), as was the case with the two known substrates. including v-Src but did not bind to neuronal specific Src in Using SU6656, a potent inhibitor of c-Src kinase, we have vitro.36 shown the involvement of three novel proteins RBM10, EWS1 We verified if the above c-Src substrates were also substrates and BTF as c-Src substrates in PDGF signaling (Figure 5C). c-Src of c-Src in vivo by cotransfecting these proteins with wild-type has been shown to have been involved in the regulation of c-Src in 293T cells. We also subcloned these cDNAs into a Flag nuclear proteins and transcription factors downstream of PDGF epitope-tagged vector, pCMVtag4A, and cotransfected 293T signaling and plays an important role in controlling DNA cells with wild-type c-Src kinase or with an empty vector. The synthesis.39 We have also examined the involvement of RasGAP proteins were immunoprecipitated using anti-Flag antibodies, SH3-domain binding protein and Thyroid hormone receptor resolved by SDS-PAGE, and immunoblotted with anti-phos- associated protein 3 in PDGF signaling using SU6656 but could photyrosine antibodies. Upon cotransfection with c-Src kinase, not detect any tyrosine phosphorylation of these proteins in we again observed increased tyrosine phosphorylation of all any state (data not shown). Further experiments need to be the tested proteins suggesting that these proteins were also in done in order to examine the precise role of these proteins and vivo substrates of c-Src (Figure 5B). We have previously the importance of their phosphorylation in PDGF signaling. identified NICE-4 as a tyrosine-phosphorylated protein in a One important caveat of these experiments using SU6656 for global phosphoproteomic study of HeLa cells.10 RBM10, FBP1 inhibition of Src kinase is that it could bind and inhibit other and TRK-fused gene are novel tyrosine-phosphorylated proteins related kinases. When better inhibitors become available, this and further investigations need to be carried out to determine would be a great approach to identify the kinase-substrate how they transduce signals downstream of c-Src kinase and if relationships. tyrosine phosphorylation regulates this process. Ewing sarcoma breakpoint region 1 (EWS1) is an RNA Involvement of a Subset of Novel Substrates in Platelet- binding protein and has been shown to be involved in gene Derived Growth Factor Signaling. Since kinase activity of c-Src translocations and often appear as fusion of EWS with ETS is required for modulating cellular responses to PDGF receptor family transcription factor genes and known to cause Ewing stimulation,37 we chose to study the role of a subset of novel sarcoma tumors.40 One of the fusion proteins EWS-Fli1 has substrates in PDGF signaling. The role of c-Src has been well- been implicated in insulin-like growth factor 1 (IGF-1)41 as well studied in PDGF signaling.38 We investigated the involvement as PDGF-BB42 induced proliferation of Ewing sarcoma cells. of EWS1, BTF and RBM10 in PDGF receptor signaling. Cortactin The implication of EWS1 as a downstream substrate of c-Src and p130CAS were used as positive controls. For this experi- in PDGF-signaling might shed light into the mechanism of ment, NIH3T3 cells, which express endogenous PDGF recep- induction of proliferation by these genes in signaling and tors, were treated with PDGF-BB in the presence or absence tumors. Bcl-2-associated transcription factor 1 (BTF) is an

Journal of Proteome Research • Vol. 7, No. 9, 2008 3905 research articles Amanchy et al.

Figure 3. MS spectra of 6 proteins identified as Src substrates by SILAC. The 3 spectral peaks in each figure represent the mass shift of the same peptide. The relative increase in intensity ratios between light to heavy are represented below in parentheses. (A) A doubly charged peptide from cortactin (1:7); (B) a triply charged peptide from NICE-4 (1:7); (C) a doubly charged peptide from Bcl2- associated transcription factor (1:7.5); (D) a doubly charged peptide from FUSE-binding protein 1 (1:8); (E) a doubly charged peptide from FUSE-binding protein 2 (1:4); (F) a doubly charged peptide from RNA binding motif 10 (1:7.5). apoptotic transcriptional repressor43 localized to the nucleus, of these proteins to show that these are indeed substrates; and whose function is still under investigation. RNA binding motif (ii) To validate these proteins in a specific signaling pathway, 10 (RBM10) is an RNA binding protein with a zinc finger it is not easy to predict in which signaling pathway(s) these domain, associated with the expression of Bax family members proteins are involved downstream of c-Src. However, studies in breast cancers and VEGF.44 This was the first time BTF and are currently in progress on a subset of proteins to show their RBM10 were identified as a tyrosine-phosphorylated proteins physiological relevance and also to identify tyrosine-phospho- and the finding that they are components of PDGF signaling rylated proteins in growth factor signaling pathways down- downstream of c-Src might help to understand the role of BTF stream of Src kinase. and RBM10 in growth factor receptor signaling. Although we Development of Peptide Microarrays for High-Throughput have observed tyrosine phosphorylation of a subset of proteins Validation of c-Src Substrates. Although we have established in PDGF signaling, it is not possible to validate all candidates above that a number of novel proteins are potential substrates in any proteomics experiment because of the following reasons: of c-Src, the exact residues that undergo phosphorylation have (i) Availability of good immunoprecipitating antibodies against not been identified in most of these instances. Hence, we these proteins is limited, and hence, we have tagged a subset developed a custom peptide microarray as a platform to rapidly

3906 Journal of Proteome Research • Vol. 7, No. 9, 2008 Identiﬁcation of c-Src Kinase Substrates research articles

Figure 4. MS/MS spectra of novel phosphorylation sites identiﬁed in this study. (A) Phosphopeptide NASTFEDVTQVSSApYQK derived from Cortactin; (B) phosphopeptide TDpYNASVSVPDSSGPER derived from hnRNPK; (C) phosphopeptide QDHPSSMGVpYGQESGG- FSGPGENR derived from Ewing sarcoma breakpoint region 1; (D) phosphopeptide IGGDAGTSLNSNDpYGYGGQK derived from FUSE- binding protein 1; (E) phosphopeptide GPSpYGLSAEVK derived from Calponin-3; (F) phosphopeptide TGAPQpYGSYGTAPVNLNIK derived from FIP1-like 1. identify the phosphopeptides on these proteins which are In all, 624 WT or mutant (312 WT and 312 MUT) peptides from phosphorylated by c-Src. We systematically designed 312 14 proteins were spotted with each sequence being represented peptides encompassing all tyrosines from 14 selected proteins in triplicate, on to the glass slides as described earlier.18 The design (Table 3). These were synthesized in such a fashion that they of these peptide microarrays is analogous to DNA microarrays contained the tyrosine residue being tested in the center. In manufactured by Affymetrix for mRNA expression studies. c-Src parallel, an equal number of peptides that have the centric kinase assays were performed on the peptide microarrays and tyrosine residues mutated to phenylalanine were designed. the arrays subsequently exposed to phosphorimager screen

Journal of Proteome Research • Vol. 7, No. 9, 2008 3907 research articles Amanchy et al. Table 3. Peptides from the Newly Identiﬁed Src Substrates That Were Phosphorylated by c-Src on Peptide Microarrays

accession no. protein phosphopeptide 1 NP_003893 FUSE binding protein 1 1. EVRNEYGSRIG 2. RQQAAYYAQTS 2 NP_003676 FUSE binding protein 2 1. GDRNEYGSRIG 2. AYYSHYYQQPP 3. RQQAAYYGQTP 3 NP_055554 BCL2-associated transcription 1. YHRGGYRPVWN factor 1 2. EETEDYRQFRK 3. GRGRGYYQGGG 4. NGSSRYSPSQN 5. RGRGYYQGGGG 6. RSSSPYSKSPV 4 NP_005667 RNA binding motif protein 10 1. ARGSSYGVTST 2. EPPVDYSYYQQ 3. DRTGRYGATDR 5 NP_006061 TRK-fused Gene 1. NEDITYDELVL 2. QMYQQYQQQAG 6 NP_005745 RasGAP SH3-domain-binding 1. NDIFRYQDEVF protein 7 NP_663760 Ataxin 2 related protein 1. GQQGKYRGAKG 8 NP_005849 A-Kinase anchor protein 8 1. RPSYSYDYEFD 9 NP_005110 Thyroid hormone receptor 1. NYRQAYSPRRG associated protein 3 2. NHPRVYQNRDF 3. SGGAAYTKRYL 4. GGYGNYRSNWQ 5. GTPAGYGRGRE 10 NP_877952 Arsenate resistance protein 1. AGRGNYDAFRG ARS2 2. QGLMPYGQPRP 3. HSSDPYHSGYE 4. KRYNDYKLDFR 11 NP_006833 Splicing factor 3B subunit 2 1. HGDLYYEGKEF 2. EEPEIYEPNFI 3. QREESYSRMGY 12 NP_055983 hypothetical protein LOC23211 1. GRGRGYRGRGS 2. DRYNSYNRPRP 3. RKYREYSPPYA

were not phosphorylated, and that the variation of WT-MUT intensities for those points was representative of nonphosphorylated peptides (Figure 6C). On the basis of the calculated FPRs, a line was drawn which separates the true positives from the remainder of the peptides. Figure 5. Experimental validation of tyrosine phosphorylation of From this analysis, we have identified phosphorylation sites proteins obtained from c-Src kinase overexpression in 293T cells. (A) In vitro kinase assays using GST tagged proteins and c-Src on 12 out of 14 proteins that were spotted on peptide microarrays. using a rabbit reticulocyte in vitro transcription and translation Peptides containing multiple tyrosines were mutated systemati- system. (B) 293T cells were cotransfected with genes of interest cally and all the tyrosine containing peptides were looked at along along with either empty vector PCMVtag4A or with c-Src. Culture with their corresponding mutated counterparts to deduce the media was changed 12 h after transfection and cells were serum- correct phosphorylated peptide. A total of 34 peptides from 12 starved for 12 h and lysed 48 h after transfection. Proteins were proteins (Table 3) out of 312 peptides from 14 proteins spotted immunoprecipitated using anti-Flag antibodies and Western (Supplementary Table 2) were phosphorylated by c-Src in our blotting was performed using phosphotyrosine antibodies and analysis. This included 6 phosphopeptides from Bcl2-associated reprobed. (C) Validation of a subset of proteins in PDGF signaling. transcription factor, 5 from Thyroid hormone receptor associated NIH3T3 cells have been grown to confluence and serum-starved protein 3, and 4 phosphopeptides from ARS2. We did not detect for 12 h followed by stimulation with PDGF-BB (100 ng/mL for 5 phosphorylation on any of the peptides derived from two proteins, min) and PDGF stimulation after treatment with SU6656 (2 µM for 1 h prior to lysis or stimulation), and cell lysates were Zinc finger, CCHC domain containing 8 and Splicing factor subjected to immunoprecipitation using anti-phosphotyrosine proline/glutamine-rich. Thus, peptide arrays allowed assignment antibodies, probed with respective antibodies, and reprobed in of phosphorylation sites in a high-throughput fashion and also whole cell lysates. served as an additional validation step for potential substrates identified from our SILAC experiments. Because of the tremen- (Figure 6A). Intensity values were obtained using GenePix software dous potential and promise that peptide microarrays hold for the - as described under Experimental Procedures. We normalized the identification kinase substrate identification, studies are ongoing intensity values and compared the log 2 intensities of the WT to spot the peptides from other proteins identified in this and peptides against their corresponding MUT peptides. The intensity other mass spectrometry based studies to identify the kinase- values from 3 different experiments were averaged individually specific phosphorylation sites and build phosphorylation motifs. for WT and MUT peptides and plotted (Figure 5B). The spots in Conclusions the upper right quadrant were taken as true positives (Figure 6B). The false positive rates (FPR) were calculated by assuming that This study describes identification of substrates of a nonre- those peptides for which MUT intensity exceeded WT intensity ceptor tyrosine kinase using a combination of proteomic

3908 Journal of Proteome Research • Vol. 7, No. 9, 2008 Identification of c-Src Kinase Substrates research articles tides from these 12 new substrates also provides validation of this approach. It is worth noting here that most tyrosine containing peptides were not phosphorylated by c-Src. Peptide microarray technology has its own limitations. Although it can help identify bona fide substrate peptides in many instances, it is possible that some sequences that are phosphorylated in vivo do not get phosphorylated because of lack of secondary and tertiary structure of the immobilized peptides. Further, although the peptide microarray analysis has shed light on the phosphopeptides preferentially phosphorylated by c-Src, these sites still remain to be investigated in vivo using other methodologies. The present study offers many encouraging leads, such as the identification of tyrosine phosphorylation of a subset of new c-Src substrates that are components in the PDGF signaling downstream of c-Src. Clearly, further characterization of these novel sites and proteins will result in a significant expansion of our knowledge of the c-Src kinase signaling network. The significance of tyrosine phosphorylation on each of the newly discovered sites remains to be determined. In any case, these results demonstrate that c-Src-mediated tyrosine phosphorylation is extensive and implicates a number of hitherto unrecognized proteins as c-Src kinase substrates.

Acknowledgment. A.P. is supported by grants from the National Institutes of Health (CA106424 and U54 RR020839), Department of Defense Era of Hope Scholar award (W81XWH-06-1-0428) and by the Beckman Young Investigator award. L.C. is supported by grants from the Figure 6. (A) Peptide microarrays: in vitro kinase assays per- National Cancer Institute (P30 CA06973-44). We thank formed on peptide microarrays, where all tyrosine containing members of Pandey lab for fruitful discussions. We also peptides and their corresponding Y f F mutant counterparts thank Hopkins Expressionists Working Group for advice and were spotted on glass slides. A representative section of the peptide microarray is magnified to show the signal correspond- comments on peptide microarray analysis. Jos Joore is VP of ing to a peptide and its Y f F mutant. (B) A classical MvA plot Array Technology at PepScan Systems. We also thank displaying data pertaining phosphorylation intensities on peptide Jurriaan Tuynman and Maikel Peppelenbosch, Academic microarrays. The horizontal dotted lines indicate 2-fold difference Medical Center, Amsterdam, The Netherlands, for their between WT and MUT intensity values. The vertical dotted line helpful suggestions on peptide microarrays. corresponds to a local false positive rate of 0.15. M on the Y-axis represents differential of (log 2 WT - log 2 MUT intensity values Supporting Information Available: This material is for each peptide and A on the X-axis represents average available free of charge via the Internet at http://pubs.acs.org. intensities ([log 2 WT + log 2 MUT]/2). (C) A plot displaying classical and local false positive rates (FPR). The red line represents local false positive rate curve and the blue line References represents the classical false positive rate. The vertical dotted (1) Thomas, S. M.; Brugge, J. S. Cellular functions regulated by Src line shows where the local FPR ) 0.15. All peptides to the right family kinases. Annu. Rev. Cell Dev. Biol. 1997, 13, 513–609. of the vertical dotted line and above 2-fold (horizontal line) were (2) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, selected as true positives. P.; Mann, M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127, 635–648. (3) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphop- approaches. Use of SILAC methodology allowed us to identify roteome analysis by mass spectrometry and its application to a number of known and novel substrates of c-Src in human Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20, 301–305. embryonic kidney 293T cells. We identified 4 new phospho- (4) Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A rylation sites and also validated a subset of the novel substrates probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 2006, as direct c-Src substrates using in vitro kinase assays. We 24, 1285–1292. corroborated our results for four proteins as direct substrates (5) Molina, H.; Horn, D. M.; Tang, N.; Mathivanan, S.; Pandey, A. of c-Src. We also implicated three of the novel c-Src substrates Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. as tyrosine-phosphorylated proteins in PDGF receptor signal- Sci. U.S.A. 2007, 104, 2199–2204. ing. Since identification of phosphopeptides is still a challenge (6) Shah, K.; Shokat, K. M. A chemical genetic screen for direct v-Src in proteomics, we designed a custom peptide microarray substrates reveals ordered assembly of a retrograde signaling platform for high-throughput identification of peptides phos- pathway. Chem. Biol. 2002, 9, 35–47. (7) Lock, P.; Abram, C. L.; Gibson, T.; Courtneidge, S. A. A new method phorylated by specific kinase, c-Src, in this case. for isolating tyrosine kinase substrates used to identify fish, an Using peptide microarrays, we identified 34 phosphopep- SH3 and PX domain-containing protein, and Src substrate. EMBO tides phosphorylated by c-Src that are derived from 12 novel J. 1998, 17, 4346–4357. (8) Qiao, Y.; Molina, H.; Pandey, A.; Zhang, J.; Cole, P. A. Chemical candidate substrate proteins that were identified by SILAC as rescue of a mutant enzyme in living cells. Science 2006, 311, 1293– potential c-Src substrates. Identification of the phosphopep- 1297.

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